scholarly journals CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1649
Author(s):  
Hao Cheng ◽  
Feng Zhang ◽  
Yang Ding
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Biomedical Applications ◽  
Viral Vectors ◽  
Recent Progress ◽  
Disease Modeling ◽  
Gene Correction ◽  
Cas9 Protein ◽  
Clinical Potential ◽  
Delivery Technology

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.

Genome editing can now be carried out in an isogenic setting. It can be effectively transmitted to somatic tissues in mice, but not to humans. Despite these doubts, CRIS has great potential as a medical promise.

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Cell Types ◽  
Polymorphic Ventricular Tachycardia ◽  
Cardiovascular Research ◽  
Cas9 Protein ◽  
Somatic Tissues ◽  
Somatic Gene ◽  
Delivery Mechanism ◽  
Editing Enzyme

As a result of genome editing, the field of cardiovascular research and treatmentis changing. Related variants can now be introduced into patient-derived cellsand tested in an isogenic environment. To evaluate the safety and efficacy ofgene-editing therapeutics, cellular phenotyping of genome-edited iPSC-derivedcardiomyocytes and other cell types will be invaluable. Parallel to theseadvances, viral vectors and nanoparticles can be used to efficiently edit genes inthe liver and core. Somatic gene editing has been used to treathypercholesterolemia, hypertriglyceridemia, WPW syndrome,catecholaminergic polymorphic ventricular tachycardia, and Duchennemuscular dystrophy in animal models. While these early achievements arepromising, they have also shown major challenges. Off-target editing withCRISPR/Cas systems will be influenced by the same editing enzyme,architecture, target cell type, and delivery mechanism. It is possible to transmitefficiently to somatic tissues in mice, but not all delivery pathways scale well tohumans. On-target unintentional editing operations, such as major deletions andinsertions, require further analysis and risk evaluation. Immunity, as well as thesubsequent development of an immune response to the Cas9 protein extractedfrom bacteria, will necessitate caution. Despite these reservations, CRISPR/Cashas tremendous therapeutic promise, and it is expected to enhance research andmedical treatment in the future.

scholarly journals Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

2019 ◽  
Author(s):  
Jacob Lamberth ◽  
Laura Daley ◽  
Pachai Natarajan ◽  
Stanislav Khoruzhenko ◽  
Nurit Becker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genome Editing ◽  
Plasmid Dna ◽  
High Efficiency ◽  
High Specificity ◽  
Human Cells ◽  
Dna And Rna ◽  
Genome Modification ◽  
Delivery Technology ◽  
Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

Genome editing of hPSCs: Recent progress in hPSC-based disease modeling for understanding disease mechanisms

2021 ◽  
Author(s):  
Dong-Kyu Choi ◽  
Yong-Kyu Kim ◽  
Ji HoonYu ◽  
Sang-Hyun Min ◽  
Sang-Wook Park
Keyword(s):  
Genome Editing ◽  
Recent Progress ◽  
Disease Modeling ◽  
Disease Mechanisms

scholarly journals Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

2018 ◽  
Author(s):  
Renata M. Martin ◽  
Kazuya Ikeda ◽  
Nobuko Uchida ◽  
Kyle Cromer ◽  
Toshi Nishimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Guide Rna ◽  
Large Gene ◽  
Marker Selection ◽  
Cas9 Protein

AbstractCombination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

scholarly journals Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

2021 ◽  
Vol 10 (14) ◽  
pp. 3141
Author(s):  
Hyerin Jung ◽  
Yeri Alice Rim ◽  
Narae Park ◽  
Yoojun Nam ◽  
Ji Hyeon Ju
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Disease Modeling ◽  
Bone Fragility ◽  
Osteogenic Potential ◽  
Type I ◽  
Gene Correction ◽  
Col1a1 Gene ◽  
Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

scholarly journals Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

2021 ◽  
Vol 22 (14) ◽  
pp. 7456
Author(s):  
Mousa A. Alghuthaymi ◽  
Aftab Ahmad ◽  
Zulqurnain Khan ◽  
Sultan Habibullah Khan ◽  
Farah K. Ahmed ◽  
...  
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Polymeric Materials ◽  
Inorganic Nanoparticles ◽  
Plant Genome ◽  
Delivery Methods ◽  
Detailed Consideration ◽  
Crispr System ◽  
Efficient Delivery ◽  
Low Cytotoxicity

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

Recent progress in carbon-dots-based nanozymes for chemosensing and biomedical applications

2021 ◽  
Author(s):  
Deming He ◽  
Minmin Yan ◽  
Pengjuan Sun ◽  
Yuanqiang Sun ◽  
Lingbo Qu ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Biomedical Applications ◽  
Recent Progress

Polydopamine antibacterial materials

2021 ◽  
Author(s):  
Yu Fu ◽  
Lei Yang ◽  
Jianhua Zhang ◽  
Junfei Hu ◽  
Gaigai Duan ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Recent Progress ◽  
Antibacterial Materials ◽  
Functional Features

This review focuses on the recent progress in polydopamine antibacterial materials, including their structural and functional features, preparation strategies, antibacterial mechanisms, and their biomedical applications.

Biomedical applications of methionine-based systems

2021 ◽  
Author(s):  
Jie Liu ◽  
Jun Huang ◽  
Peikun Xin ◽  
Guiting Liu ◽  
Jun Wu
Keyword(s):  
Liver Disease ◽  
Cancer Treatment ◽  
Biomedical Applications ◽  
Recent Progress ◽  
Functional Modification ◽  
Disease Therapy ◽  
Derivatives Of

Spurred by the structure, metabolism, and derivatives of methionine, this review systematically summarizes its recent progress in functional modification, cancer treatment, liver disease therapy.

scholarly journals Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pan P. Li ◽  
Russell L. Margolis
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Neurodegenerative Disorder ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
End Joining ◽  
Human Ipscs ◽  
Non Homologous End Joining ◽  
Transient Overexpression ◽  
Donor Template

AbstractCas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.

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